Salt and crystal forms of PLK-4 inhibitor

ABSTRACT

A fumarate salt and a maleate salt of compound (I) represented by the following structural formula, as well as their corresponding pharmaceutical compositions, are disclosed. Particular single crystalline forms of 1:1 compound (I) fumarate and 1:1 compound (I) maleate are characterized by a variety of properties and physical measurements. Methods of preparing specific crystalline forms of 1:1 compound (I) fumarate and 1:1 compound (I) maleate are also disclosed. The present invention also provides methods of treating a subject with a cancer. Compound

RELATED APPLICATIONS

This application is a U.S. national stage application of InternationalApplication No. PCT/CA2014/051001, filed Oct. 17, 2014, which claims thebenefit of U.S. Provisional Application No. 61/892,564, filed Oct. 18,2013. The entire teachings of each of the aforementioned applicationsare incorporated herein by reference.

BACKGROUND

The polo-like kinase (PLK) family of serine/threonine kinases comprisesat least four known members: PLK1, PLK2 (also known as Snk), PLK3 (alsoknown as Fnk or Prk) and PLK4 (also known as Sak). Agents which inhibitPLK4 have the potential to treat cancer. A number of potent PLK4inhibitors are disclosed in U.S. Pat. Nos. 8,263,596, 8,481,525, and8,481,533 (the entire teachings of which are incorporated herein byreference). The structure of one inhibitor disclosed in these patents isshown below as compound (I):

There is a need for salt forms of this compound that are crystalline andotherwise have physical properties that are amenable to large scalemanufacture. There is also a need for pharmaceutical formulations inwhich this drug candidate is stable and is effectively delivered to thepatient.

SUMMARY OF THE INVENTION

It has been found that the 1:1 fumaric acid salt and the 1:1 maleic acidsalt of compound (I) can be crystallized under well-defined conditionsto provide non-hygroscopic crystalline forms. The designation “1:1” isthe molar ratio between acid (fumaric or maleic) and compound (I).Because of the two carboxylic acid groups on fumaric acid and maleicacid, it is also possible to form a 1:2 fumaric acid salt and a 1:2maleic acid salt of compound (I), in which the molar ratio between acid(fumaric or maleic) and compound (I) is 1:2. The 1:1 fumaric acid saltof compound (I) is referred to herein as “1:1 compound (I) fumarate”;and the 1:1 maleic acid salt is referred to herein as “1:1 compound (I)maleate”.

1:1 Compound (I) fumarate and 1:1 compound (I) maleate have severaladvantageous properties when compared with other salts of compound (I).As shown in Examples 1 and 2, many salts of compound (I), includinghydrochloride salt, phosphate, sulfate, and citrate, could not beobtained in crystal form. Notably, 1:2 compound (I) fumarate and 1:2compound (I) maleate also could not be obtained in crystalline form. 1:1Compound (I) fumarate and 1:1 compound (I) maleate are bothnon-hygroscopic and easier to formulate than the free base and the othersalts. Thus, these favorable properties make 1:1 compound (I) fumarateand 1:1 compound (I) maleate amenable to large scale manufacture andformulation as a drug candidate.

In one aspect, the present invention provides a fumarate salt ofcompound (I) wherein the molar ratio between compound (I) and fumaricacid is 1:1. As noted above, this salt is also referred to herein as“1:1 compound (I) fumarate”.

In another aspect, the present invention provides a maleate salt ofcompound (I) wherein the molar ratio between compound (I) and maleicacid is 1:1. As noted above, this salt is also referred to herein as“1:1 compound (I) maleate”.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising 1:1 compound (I) fumarate (or 1:1 compound (I)maleate) and a pharmaceutically acceptable carrier or diluent.

In still another aspect, the present invention provides a method oftreating a subject with cancer, comprising administering to the subjectan effective amount of 1:1 compound (I) fumarate or 1:1 compound (I)maleate.

The present invention provides a method of inhibiting PLK4 activity in asubject in need of inhibition of PLK4 activity, comprising administeringto the subject an effective amount of 1:1 compound (I) fumarate or 1:1compound (I) maleate.

The present invention also provides 1:1 compound (I) fumarate or 1:1compound (I) maleate for use in medicinal therapy. In one embodiment,the medicinal therapy is for treating a subject with cancer.Alternatively, the therapy is for inhibiting PLK4 activity in a subjectin need of inhibition of PLK4 activity.

Another aspect of the present invention is the use of 1:1 compound (I)fumarate or 1:1 compound (I) maleate for the manufacture of a medicamentfor treating a subject with cancer.

Another aspect of the present invention is 1:1 compound (I) fumarate or1:1 compound (I) maleate for treating a subject with cancer.

Another aspect of the present invention is the use of a 1:1 compound (I)fumarate or 1:1 compound (I) maleate for the manufacture of a medicamentfor inhibiting PLK4 activity in a subject in need of inhibition of PLK4activity.

Another aspect of the present invention is 1:1 compound (I) fumarate or1:1 compound (I) maleate for inhibiting PLK4 activity in a subject inneed of inhibition of PLK4 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Differential Scanning calorimetry Analysis (DSC)thermogram for form A of 1:1 compound (I) fumarate.

FIG. 2 shows the DSC thermogram for form B of 1:1 compound (I) fumarate.

FIG. 3 shows the DSC thermogram for form C of 1:1 compound (I) fumarate.

FIG. 4 shows the DSC thermogram for form D of 1:1 compound (I) fumarate.

FIG. 5 shows the DSC thermogram for form A of 1:1 compound (I) maleate.

FIG. 6 shows the DSC thermogram for phosphate of compound (I).

FIG. 7 shows the X-ray Powder Diffraction (XRPD) pattern for form A of1:1 compound (I) fumarate.

FIG. 8 shows the XRPD pattern for form B of 1:1 compound (I) fumarate.

FIG. 9 shows the XRPD pattern for form C of 1:1 compound (I) fumarate.

FIG. 10 shows the XRPD pattern for form D of 1:1 compound (I) fumarate.

FIG. 11 shows the XRPD pattern for form A of 1:1 compound (I) maleate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides 1:1 compound (I) fumarate, 1:1 compound(I) maleate, unique crystalline forms thereof and their correspondingpharmaceutical compositions. The present invention also provides methodsof treating a subject with a cancer. Additionally, the present inventionprovides methods for preparing specific crystalline forms of 1:1compound (I) fumarate and 1:1 compound (I) maleate.

Crystalline Forms of 1:1 Compound (I) Fumarate and 1:1 Compound (I)Maleate

In a particular embodiment, at least a particular percentage by weightof 1:1 compound (I) fumarate or 1:1 compound (I) maleate is crystalline.Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.9%, or a weight percentage of 70%-75%, 75%-80%, 80%-85%,85%-90%, 90%-95%, 95%-100%, 70-80%, 80-90%, 90-100%. For example, in oneembodiment, at least 80% (e.g., at least 90% or 99%) by weight of the1:1 compound (I) fumarate or the 1:1 compound (I) maleate iscrystalline. It is to be understood that all values and ranges betweenthese values and ranges are meant to be encompassed by the presentinvention.

In another particular embodiment, at least a particular percentage byweight of 1:1 compound (I) fumarate and 1:1 compound (I) maleate is asingle crystalline form. Particular weight percentages include 70%, 72%,75%, 77%, 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or a weight percentage of70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, 95%-100%, 70-80%, 80-90%,90-100%. For example, in one embodiment, at least 80% (e.g., at least90% or 99%) by weight of the 1:1 compound (I) fumarate and 1:1 compound(I) maleate is in a single crystalline form. It is to be understood thatall values and ranges between these values and ranges are meant to beencompassed by the present invention.

As used herein, “crystalline” refers to a solid having a crystalstructure wherein the individual molecules have a highly homogeneousregular locked-in chemical configuration. Crystalline 1:1 compound (I)fumarate and crystalline 1:1 compound (I) maleate can be crystals of asingle crystalline form of 1:1 compound (I) fumarate and 1:1 compound(I) maleate, or a mixture of crystals of different single crystallineforms. A single crystalline form means 1:1 compound (I) fumarate or 1:1compound (I) maleate as a single crystal or a plurality of crystals inwhich each crystal has the same crystal form.

When a particular percentage by weight of 1:1 compound (I) fumarate (or1:1 compound (I) maleate) is a single crystalline form, the remainder ofthe fumarate (or 1:1 compound (I) maleate) is some combination ofamorphous fumarate (or 1:1 compound (I) maleate), and/or one or moreother crystalline forms of 1:1 compound (I) fumarate (or 1:1 compound(I) maleate) excluding the single crystalline form. When the crystalline1:1 compound (I) fumarate (or crystalline 1:1 compound (I) maleate) isdefined as a specified percentage of one particular crystalline form of1:1 compound (I) fumarate (or 1:1 compound (I) maleate), the remainderis made up of amorphous form and/or crystalline forms other than the oneor more particular forms that are specified. Examples of a singlecrystalline form include form A of 1:1 compound (I) fumarate (or 1:1compound (I) maleate) characterized by one or more properties asdiscussed herein.

1:1 Compound (I) fumarate (or 1:1 compound (I) maleate) is at least 60%,70%, 80%, 90%, 99% or 99.9% by weight pure relative to the otherstereoisomers, i.e., the ratio of the weight of the stereoisomer overthe weight of all the stereoisomers.

Preparation of Crystalline Forms of 1:1 Compound (I) Fumarate and 1:1Compound (I) Maleate

The particular solid forms of 1:1 compound (I) fumarate or 1:1 compound(I) maleate can be prepared, for example, by slow evaporation, slowcooling, and antisolvent precipitation.

As used herein, “anti-solvent” refers to a solvent, in which 1:1compound (I) fumarate or 1:1 compound (I) maleate has low solubility andcause the fumarate or maleate to precipitate out of solution in the formof fine powder or crystals.

Alternatively, 1:1 compound (I) fumarate or 1:1 compound (I) maleate canthen be recrystallized from a suitable solvent with or without theaddition of a seed crystal.

The preparation of each specific solid forms of 1:1 compound (I)fumarate or 1:1 compound (I) maleate is described in the experimentalsection below.

Characterization of Crystalline Forms of 1:1 Compound (I) Fumarate and1:1 Compound (I) Maleate

Samples are irradiated with copper K-alpha X-rays with the X-ray tubeoperated at 40 kV/30 mA. In one embodiment, 1:1 compound (I) fumarate isa single crystalline form, form A. In a specific embodiment, form A of1:1 compound (I) fumarate is characterized by the X-ray powderdiffraction pattern shown in FIG. 7. In a more particular embodiment,1:1 compound (I) fumarate form A is characterized by an X-ray powderdiffraction pattern which comprises peaks (2θ angles) at:

a) 9.7°, 16.7°, and 20.1°±0.2 in 2θ (major peaks); or

b) 8.2°, 9.7°, 16.7°, and 20.1°±0.2 in 2θ; or

c) 8.2°, 9.7°, 10.7°, 11.5°, 14.9°, 16.7°, 20.1°, and 23.5°±0.2 in 2θ;or

d) 8.2°, 9.7°, 10.7°, 11.5°, 13.6°, 14.9°, 16.7°, 18.1°, 18.8°, 20.1°,23.5°, and 24.5°±0.2 in 2θ.

The major peaks described herein have a relative intensity over 50% inform A. It is to be understood that a specified 2θ angle means thespecified value ±0.2°.

In another specific embodiment, 1:1 compound (I) fumarate form A ischaracterized by differential scanning calorimeter (DSC) peak phasetransition temperatures of 112° C. and 158° C.

In another embodiment, 1:1 compound (I) fumarate is a single crystallineform, form B. In a specific embodiment, form B of 1:1 compound (I)fumarate is characterized by the X-ray powder diffraction pattern shownin FIG. 8. In a more particular embodiment, 1:1 compound (I) fumarateform B is characterized by an X-ray powder diffraction pattern whichcomprises peaks at:

a) 11.9° and 14.9°±0.2 in 2θ (major peaks); or

b) 11.9°, 14.9°, 18.7°, and 21.5°±0.2 in 2θ; or

c) 5.5°, 5.9°, 11.9°, 14.9°, 16.7°, 17.4°, 18.7°, 21.5°, and 23.4°±0.2in 2θ.

The major peaks described herein have a relative intensity over 75% inform B.

In another specific embodiment, 1:1 compound (I) fumarate form B ischaracterized by differential scanning calorimeter (DSC) peak phasetransition temperatures of 58° C. and 162° C.

In another embodiment, 1:1 compound (I) fumarate is a single crystallineform, form C. In a specific embodiment, 1:1 compound (I) fumarate form Cis characterized by the X-ray powder diffraction pattern of the singlecrystalline shown in FIG. 9. In a more particular embodiment, 1:1compound (I) fumarate form C is characterized by an X-ray powderdiffraction pattern which comprises peaks at:

a) 16.8°, 16.9°, and 19.9°±0.2 in 2θ (major peaks); or

b) 9.8°, 16.8°, 16.9°, 19.9°, and 23.5°±0.2 in 2θ; or

c) 9.7°, 9.8°, 11.7°, 15.1°, 16.8°, 16.9°, 19.9°, 23.5°, and 23.7°±0.2in 2θ.

The major peaks described herein have a relative intensity over 75% inform C.

In another specific embodiment, 1:1 compound (I) fumarate form C ischaracterized by differential scanning calorimeter (DSC) peak phasetransition temperatures of 62° C. and 156° C.

In another embodiment, 1:1 compound (I) fumarate is a single crystallineform, form D. In a specific embodiment, Form D of 1:1 compound (I)fumarate is characterized by the X-ray powder diffraction pattern inFIG. 10. In a more specific embodiment, the X-ray diffraction pattern ofForm D comprises peaks at:

a) 9.6°, 12.8°, 16.0°, and 22.0°±0.2 in 2θ (major peaks); or

b) 9.6°, 12.8°, 16.0°, 16.9°, 21.2°, and 22.0°±0.2 in 2θ; or

c) 9.6°, 12.8°, 16.0°, 16.9°, 20.8°, 21.2°, 21.5°, and 22.0°±0.2 in 2θ.

d) 9.6°, 11.7°, 12.0°, 12.8°, 16.0°, 16.6°, 16.9°, 18.1°, 19.2°, 19.8°,20.7°, 20.8°, 21.2°, 21.5°, 22.0°, 22.5°, 24.0°, 26.0°, and 29.8°±0.2 in2θ.

The major peaks described herein have a relative intensity over 85% inform D.

In another specific embodiment, at least 90% by weight of the 1:1compound (I) fumarate Form D is characterized by differential scanningcalorimeter (DSC) peak phase transition temperature of 219° C.

In another embodiment, 1:1 compound (I) maleate is a single crystallineform, Form A. In a specific embodiment, form A of 1:1 compound (I)maleate is characterized by the X-ray powder diffraction pattern in FIG.11. In a more specific embodiment, form A of 1:1 compound (I) maleate ischaracterized by the X-ray powder diffraction pattern which comprisespeaks at:

a) 11.5°, 12.6°, 14.9°, and 15.1°±0.2 in 2θ; or

b) 10.8°, 11.5°, 12.4°, 12.6°, 14.9°, 15.1°, and 17.1°±0.2 in 2θ; or

c) 5.8°, 10.8°, 11.5°, 12.4°, 12.6°, 14.9°, 15.1°, 17.1°, 18.6°, 23.5°,and 26.1°±0.2 in 2θ; or

d) 5.5°, 5.8°, 10.8°, 11.5°, 12.4°, 12.6°, 14.1°, 14.9°, 15.1°, 16.7°,17.1°, 17.8°, 18.6°, 19.5°, 19.9°, 21.9°, 22.2°, 23.0°, 23.3°, 23.5°,23.9°, and 26.1°±0.2 in 2θ.

The major peaks described herein have a relative intensity over 90% inform A.

In another specific embodiment, 1:1 compound (I) maleate form A ischaracterized by differential scanning calorimeter (DSC) peak phasetransition temperature of 219° C.

The fumarate salt of compound (I) or the maleate salt of compound (I)described herein is either in an amorphous form or in a crystallineform. The fumarate salt of compound (I) or the maleate salt of compound(I) described in the present invention includes both an unsolvated formand a solvate form.

“Solvate form” refers to a solid or a crystalline form of the fumaratesalt of compound (I) or the maleate salt of compound (I), where solventis combined with the fumarate salt of compound (I) or the maleate saltof compound (I) in a definite ratio (e.g., a molar ratio of 1:1 or 1:2)as an integral part of the solid or a crystal.

“Unsolvated form” refers to no definite ratio between a solvent moleculeand the fumarate salt of compound (I) or the maleate salt of compound(I), and the solvent molecule is not substantially (e.g., less that 10%by weight) existed in the fumarate salt of compound (I) or the maleatesalt of compound (I). Well known solvent molecules include water,methanol, ethanol, n-propanol, and isopropanol.

In the present invention, form A of 1:1 compound (I) fumarate is anisopropanol solvate, which has a molar ratio of 2:1 between the compound(I) fumarate and isopropanol. Form B-D of 1:1 compound (I) fumarate andform A of 1:1 compound (I) maleate described herein are not solvates,i.e., each are an unsolvated form.

Methods of Treatment Using Compound (I) Fumarate and Compound (I)Maleate

1:1 Compound (I) fumarate and 1:1 compound (I) maleate can inhibitvarious kinases, including PLK4. Thus, 1:1 compound (I) fumarate and 1:1compound (I) maleate of the invention are useful in the treatment ofdiseases or conditions associated with such kinase. For example, PLK4 isbelieved to be involved in cellular mitotic progression. Thus, smallmolecule inhibitors of this enzyme can be potential anti-tumor agents.

In a specific embodiment, 1:1 compound (I) fumarate and 1:1 compound (I)maleate are PLK4 inhibitors, and are useful for treating diseases, suchas cancer, associated with such a kinase.

Another aspect of the invention relates to a method of treating asubject with cancer, comprising administering to the subject aneffective amount of 1:1 compound (I) fumarate and 1:1 compound (I)maleate. In one embodiment, 1:1 compound (I) fumarate and 1:1 compound(I) maleate inhibit the growth of a tumor. Specifically, 1:1 compound(I) fumarate and 1:1 compound (I) maleate inhibit the growth of a tumorthat overexpresses PLK4. In another embodiment, 1:1 compound (I)fumarate and 1:1 compound (I) maleate inhibit the growth of the tumor byinducing apoptosis of the tumor cells or by inhibiting proliferation ofthe tumor cells.

Cancers that can be treated or prevented by the methods of the presentinvention include lung cancer, breast cancer, colon cancer, braincancer, neuroblastoma, prostate cancer, melanoma, glioblastomamultiform, ovarian cancer, lymphoma, leukemia, melanoma, sarcoma,paraneoplasia, osteosarcoma, germinoma, glioma and mesothelioma. In onespecific embodiment, the cancer is lung cancer, breast cancer, coloncancer, neuroblastoma, prostate cancer, melanoma, glioblastomamultiforme, ovarian cancer, lymphoma, leukemia, osteosarcoma, germinoma,glioma, fibrosarcoma, gastrointestinal sarcoma, fibrous histiocytoma,round cell sarcoma, synovial sarcoma, cervical cancer, anogenitalcancer, head and neck cancer, and oropharyngeal cancer. In one specificembodiment, the cancer is lung cancer, colon cancer, brain cancer,neuroblastoma, prostate cancer, melanoma, glioblastoma multiforme orovarian cancer. In another specific embodiment, the cancer is lungcancer, breast cancer, colon cancer, brain cancer, neuroblastoma,prostate cancer, melanoma, glioblastoma multiform or ovarian cancer. Inanother specific embodiment, the cancer is lung cancer, breast cancer,and colon cancer. In yet another specific embodiment, the cancer is abreast cancer. In yet another specific embodiment, the cancer is a basalsub-type breast cancer or a luminal B sub-type breast cancer. In oneembodiment, the basal sub-type breast cancer is ER (estrogen receptor),HER2 and PR (progesterone receptor) negative breast cancer. In yetanother specific embodiment, the cancer is a soft tissue cancer. A “softtissue cancer” is an art-recognized term that encompasses tumors derivedfrom any soft tissue of the body. Such soft tissue connects, supports,or surrounds various structures and organs of the body, including, butnot limited to, smooth muscle, skeletal muscle, tendons, fibroustissues, fatty tissue, blood and lymph vessels, perivascular tissue,nerves, mesenchymal cells and synovial tissues. Thus, soft tissuecancers can be of fat tissue, muscle tissue, nerve tissue, joint tissue,blood vessels, lymph vessels, and fibrous tissues. Soft tissue cancerscan be benign or malignant. Generally, malignant soft tissue cancers arereferred to as sarcomas, or soft tissue sarcomas. There are many typesof soft tissue tumors, including lipoma, lipoblastoma, hibernoma,liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma,neurofibroma, schwannoma (neurilemoma), neuroma, malignant schwannoma,neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis, synovialsarcoma, hemangioma, glomus tumor, hemangiopericytoma,hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma,fibroma, elastofibroma, superficial fibromatosis, fibrous histiocytoma,fibrosarcoma, fibromatosis, dermatofibrosarcoma protuberans (DFSP),malignant fibrous histiocytoma (MFH), myxoma, granular cell tumor,malignant mesenchymomas, alveolar soft-part sarcoma, epithelioidsarcoma, clear cell sarcoma, and desmoplastic small cell tumor. In aparticular embodiment, the soft tissue cancer is a sarcoma selected fromthe group consisting of a fibrosarcoma, a gastrointestinal sarcoma, aleiomyosarcoma, a dedifferentiated liposarcoma, a pleomorphicliposarcoma, a malignant fibrous histiocytoma, a round cell sarcoma, anda synovial sarcoma.

The invention further relates to a method of treating a subject withtumor cells, comprising administering to the subject, an amount of acompound disclosed herein that is effective to reduce effectively PLK4activity in the subject.

The term an “effective amount” means an amount when administered to thesubject which results in beneficial or desired results, includingclinical results, e.g., inhibits, suppresses or reduces the cancer(e.g., as determined by clinical symptoms or the amount of cancer cells)in a subject as compared to a control.

As used herein, “treating a subject with a cancer” includes achieving,partially or substantially, one or more of the following: arresting thegrowth of a cancer, reducing the extent of a cancer (e.g., reducing sizeof a tumor), inhibiting the growth rate of a cancer, ameliorating orimproving a clinical symptom or indicator associated with the cancer(such as tissue or serum components) or increasing longevity of thesubject; and reducing the likelihood of recurrence of the cancer.

As used herein, the term “reducing the likelihood of recurrence of acancer” means inhibiting or delaying the return of a cancer at or near aprimary site and/or at a secondary site after a period of remission. Italso means that the cancer is less likely to return with treatmentdescribed herein than in its absence.

As used herein, the term “remission” refers to a state of cancer,wherein the clinical symptoms or indicators associated with a cancerhave disappeared or cannot be detected, typically after the subject hasbeen successfully treated with an anti-cancer therapy.

Generally, an effective amount of a compound of the invention variesdepending upon various factors, such as the given drug or compound, thepharmaceutical formulation, the route of administration, the type ofdisease or disorder, the identity of the subject or host being treated,and the like, but can nevertheless be routinely determined by oneskilled in the art. An effective amount of a compound of the presentinvention may be readily determined by one of ordinary skill by routinemethods known in the art.

In an embodiment, an effective amount of 1:1 compound (I) fumarate and1:1 compound (I) maleate ranges from about 0.01 to about 1000 mg/kg bodyweight, alternatively about 0.05 to about 500 mg/kg body weight,alternatively about 0.1 to about 100 mg/kg body weight, alternativelyabout 0.1 to about 15 mg/kg body weight, alternatively about 1 to about5 mg/kg body weight, and in another alternative, from about 2 to about 3mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat a subjectsuffering from cancer and these factors include, but are not limited to,the severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject and other diseases present.

Moreover, a “treatment” regime of a subject with an effective amount ofthe compound of the present invention may consist of a singleadministration, or alternatively comprise a series of applications. Forexample, 1:1 compound (I) fumarate and 1:1 compound (I) maleate may beadministered at least once a week. However, in another embodiment, thecompound may be administered to the subject from about one time per weekto once daily for a given treatment. The length of the treatment perioddepends on a variety of factors, such as the severity of the disease,the age of the patient, the concentration and the activity of thecompounds of the present invention, or a combination thereof. It willalso be appreciated that the effective dosage of the compound used forthe treatment or prophylaxis may increase or decrease over the course ofa particular treatment or prophylaxis regime. Changes in dosage mayresult and become apparent by standard diagnostic assays known in theart. In some instances, chronic administration may be required.

A “subject” is a mammal, preferably a human, but can also be an animalin need of veterinary treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, andthe like) and laboratory animals (e.g., rats, mice, guinea pigs, and thelike).

The compounds of the invention can be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The compounds of theinvention may be administered, for example, by oral, parenteral, buccal,sublingual, nasal, rectal, patch, pump or transdermal administration andthe pharmaceutical compositions formulated accordingly. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationcan be by continuous infusion over a selected period of time.

Pharmaceutical Compositions Including 1:1 Compound (I) Fumarate and 1:1Compound (I) Maleate

1:1 Compound (I) fumarate or 1:1 compound (I) maleate or any one or moreof the crystal forms disclosed herein can be suitably formulated intopharmaceutical compositions for administration to a subject.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising 1:1 Compound (I) fumarate or 1:1 compound (I)maleate as described above, and a pharmaceutically acceptable carrier ordiluent, wherein at least 80% (preferably 90%, more preferably 99%) byweight of the salt is crystalline.

The pharmaceutical compositions of the present teachings optionallyinclude one or more pharmaceutically acceptable carriers and/or diluentstherefor, such as lactose, starch, cellulose and dextrose. Otherexcipients, such as flavoring agents; sweeteners; and preservatives,such as methyl, ethyl, propyl and butyl parabens, can also be included.More complete listings of suitable excipients can be found in theHandbook of Pharmaceutical Excipients (5^(th) Ed., Pharmaceutical Press(2005)). A person skilled in the art would know how to prepareformulations suitable for various types of administration routes.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003—20th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999. The carriers, diluents and/or excipients are“acceptable” in the sense of being compatible with the other ingredientsof the pharmaceutical composition and not deleterious to the recipientthereof.

Typically, for oral therapeutic administration, a compound of thepresent teachings may be incorporated with excipient and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

Typically for parenteral administration, solutions of a compound of thepresent teachings can generally be prepared in water suitably mixed witha surfactant such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, DMSO and mixturesthereof with or without alcohol, and in oils. Under ordinary conditionsof storage and use, these preparations contain a preservative to preventthe growth of microorganisms.

Typically, for injectable use, sterile aqueous solutions or dispersionof, and sterile powders of, a compound described herein for theextemporaneous preparation of sterile injectable solutions ordispersions are appropriate.

For nasal administration, the compounds of the present teachings can beformulated as aerosols, drops, gels and powders. Aerosol formulationstypically comprise a solution or fine suspension of the active substancein a physiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant which can be a compressed gassuch as compressed air or an organic propellant such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

For buccal or sublingual administration, the compounds of the presentteachings can be formulated with a carrier such as sugar, acacia,tragacanth, or gelatin and glycerine, as tablets, lozenges or pastilles.

For rectal administration, the compounds described herein can beformulated in the form of suppositories containing a conventionalsuppository base such as cocoa butter.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXPERIMENTAL

Abbreviations:

BSA benzene sulfonic acid

d days

EtOAc ethyl acetate

EtOH ethanol

h hours

HPLC high performance liquid chromatography

IPA isopropanol

IBAc isobutyl acetate

MeOH methanol

MIBK methyl isobutyl ketone

min minutes

MTBE methyl tert-butylether

NMR nuclear magnetic resonance

pTSA para toluenesulfonic acid

RBF round bottom flask

RH relative humidity

Rel. Int. relative intensity

rt room temperature

temp temperature

TGA thermogravimetric analysis

THF tetrahydrofuran

wt % percent by weight

XRPD X-ray powder diffraction

Analysis Conditions

Differential Scanning Calorimetry Analysis (DSC)

DSC analyses were carried out on a Mettler 822e differential scanningcalorimeter or a TA instruments Q2000. Samples were weighed in analuminum pan, covered with a pierced lid, and then crimped. Analysisconditions were 30-120, 30-200, 30-300° C. and 40-300° C. ramped at 10°C./min.

Thermal Gravimetric Analysis (TGA)

TGA analyses were carried out on a Mettler 851e SDTA thermogravimetricanalyzer. Samples were weighed in an alumina crucible and analyzed from30-230, 30-300 and 30-350° C. and at a ramp rate of 10° C./min.

X-Ray Powder Diffraction (XRPD)

Samples were analyzed on a Panalytical CubiX-Pro X-ray powderdiffractometer or a Bruker AXS/Siemens D5000 diffractometer.

Panalytical Conditions: Samples were placed on a silicon zero-returnultra-micro sample holder. The samples were irradiated with copperK-alpha X-rays with the X-ray tube operated at 40 kV/30 mA. The sampleswere scanned in continuous mode along the range 3 to 45°.

Bruker AXS/Siemens D5000 Conditions: A high-power Cu-target was usedoperating at 50 kV/35 mA. The secondary beam was monochromatized by asolid state Kevex detector. The samples were scanned along the range2-35° (2σ) where representative peaks for most of the organiccrystalline compounds occur.

Gravimetric Moisture Sorption

Gravimetric moisture sorption experiments were carried out on a Hidendynamic vapour sorption analyzer by first holding the sample at 40% RHand 25° C. until an equilibrium weight was reached or for a maximum offour hours. The sample was then subjected to an isothermal (25° C.)adsorption scan from 40 to 90% RH in steps of 10%. The sample wasallowed to equilibrate to an asymptotic weight at each point for amaximum of four hours. Following adsorption, a desorption scan from 85to 5% RH (at 25° C.) was run in steps of −10% again allowing a maximumof four hours for equilibration to an asymptotic weight. An adsorptionscan was then performed from 0% RH to 40% RH in steps of +10% RH.

Raman Spectroscopy

Samples for Raman analysis were analyzed on a Kaiser RXN1 Macroscopewith PhAT Probe. Solids obtained from the 96 well plate crystallizationswere analyzed using the following conditions:

Raman Source: 785 nm laser

Microscope Objective 1.2 mm

Single Exposure Time: 12 sec

Co-additions: 12

Enabled Exposure options: Cosmic Ray filtering, Dark Subtraction,Intensity Calibration

Optical Microscopy

Samples were examined with a Leica DMRB polarized light microscopecombined with a digital camera (1600×1200 resolution). Small amounts ofsamples were dispersed in mineral oil on a glass slide with cover slipsand viewed with 100× magnification or higher.

Birefringence

Samples for Birefringence analysis were analyzed on a ColemanTechnologies birefringence imager. Solids obtained from the 96 wellplate crystallizations were analyzed using the following conditions:

Lighting: 37

Exposure: 57.9

Polarization: 0.0

Well mask diameter: 6.2

Target Intensity: 80

Target Percentile: 90.0

Maximum Mean Intensity: 100

Nuclear Magnetic Resonance

Samples for Proton NMR were analyzed using a Bruker 400 MHzspectrometer.

Example 1 Combinatorial Salt Screen

A salt screen was performed using six solvents (IPA, THF, acetone,acetonitrile, EtOH and EtOAc) and twenty-eight pharmaceuticallyacceptable acids (HCl, HBr, H₃PO₄, H₂SO₄, CH₃SO₃H, pTSA, BSA,naphthalene sulfonic acid, ethane sulfonic acid, methane sulfonic acid,adipic acid, ethane disulfonic acid, maleic acid, benzoic acid, L-malicacid, citric acid, L-lactic acid, hippuric acid, L-pyroglutamic acid,succinic acid, L-tartaric acid, formic acid, fumaric acid, glutaricacid, L-ascorbic acid, sorbic acid, benzoic acid, and malonic acid). A96-well plate was charged with 200 μL of a 20 mg/mL solution of compound(I) in MeOH in each well. The solvent was then evaporated under a flowof nitrogen, leaving approximately 4 mg of the starting material in eachwell. The primary solvent of interest was then added to each well (500μL). The plates were heated to 50° C. and stirred magnetically for 10min to ensure complete dissolution of A. Each well was then charged withthe designated counterion solution at a volume corresponding to 1.05equivalents of each acid and allowed to equilibrate at temperature for10 min. The plates were then cooled at 20° C./h to 25° C., at whichpoint the master plate was daughtered by transferring 200 μL from eachwell into an evaporation plate. The plates were then cooled to ambienttemperature, stored overnight at 5° C. and checked for the presence ofsolids. The solvent from the master plates was then removed by wickingwith sorbent paper. The master and evaporation plates were then driedunder nitrogen overnight. Wells that contained solid material were thenscored and ranked for birefringence, unique Raman spectrum and thresholdsolubility. Suitable solvent and counterion combinations were thenreevaluated for salt formation with A at an increased scale.

Example 2 Intermediate Scale Salt Formation

Approximately 40 mg of compound (I), was weighed into an 8 mL vialcontaining a magnetic stir bar. To the vial, primary solvent was addedto ensure dissolution at elevated temperature. Following dissolution,1.05 equivalents of acid was added dropwise as a 0.125, 0.25 or 0.5 Msolution. All mixtures were allowed to stir at elevated temperature for15 min, followed by cooling to room temperature at a rate of 10° C./hand stirring overnight. Samples that did not exhibit precipitation aftercooling were scratched with a spatula to induce nucleation and stored ina freezer at −10 to −20° C. The vials were inspected for crystal growthafter 1 h. Samples from conditions that afforded solids werecentrifuged, filtered or evaporated under nitrogen. All other sampleswere allowed to equilibrate at −20° C. for 72 h. Vials that did not showprecipitation were then dried under nitrogen. The resulting solids werethen slurried with IPA for 5 d. From these experiments, only amorphoussolids were identified for all counterions except for the fumaric acidsalt. Unique polymorphic forms of the fumarate salt of compound (I) aredescribed below.

Preparation of Crystalline Salts of Compound (I) Example 3 Preparationof Form A of 1:1 Compound (I) Fumarate

Compound (I) (42 mg, 0.078 mmol) was dissolved into acetonitrile (0.5mL) and heated to 50° C. Fumaric acid (0.33 mL of 0.25M solution in IPA)was added and the mixture stirred for 15 min. The precipitate wasfiltered and determined to be amorphous due to lack of observablebirefringence. The amorphous solid was then slurried with IPA (0.5 mL)for 5 d. The solid obtained from the slurry showed birefringence and wasfurther characterized as an IPA solvate by XRPD, DSC, ¹H NMR and TGA anddenoted fumarate form A.

TABLE 1 XRPD of Fumarate Form A 2θ angle Rel. Int. (%) 8.17 43% 9.69100% 10.69 33% 11.51 30% 13.63 25% 14.89 36% 16.73 61% 18.09 27% 18.8323% 20.05 50% 23.49 34% 24.45 26%

Example 4 Preparation of Form B of 1:1 Compound (I) Fumarate

Desolvation of fumarate form A by vacuum drying at 60° C. for 2 dresulted in a crystalline material with a DSC thermogram and XRPDdesignated as form B. Form B can also be prepared directly by dissolvingthe amorphous monofumarate salt in EtOAc and seeding with form Bcrystals. Gravimetric moisture sorption indicates that the salt form ishygroscopic and forms a tetrahydrate at 90% RH.

TABLE 2 XRPD of Fumarate Form B 2θ angle Rel. Int. (%) 5.51 39.5 5.9138.7 11.91 100.0 14.93 83.5 16.71 40.2 17.35 40.4 18.73 42.8 21.53 41.023.41 43.3

Example 5 Preparation of Form C of 1:1 Compound (I) Fumarate

A third fumarate polymorph can be obtained by dissolution of amorphousfumarate salt into THF and seeding with fumarate form B crystals. Thesolvent was slowly evaporated to a white solid which exhibited a XRPDpattern and Raman spectrum different from form B and was designated asfumarate form C.

TABLE 3 XRPD of Fumarate Form C Rel. 2θ angle Int. (%) 9.71 68.1 9.8370.2 11.71 59.5 15.05 60.1 16.83 88.7 16.87 84.7 19.93 100.0 23.45 70.723.65 69.6

Example 6 Preparation of Form D of 1:1 Compound (I) Fumarate

Slurrying fumarate form C with acetonitrile for 10 d at rt resulted intransition to a new crystal form, denoted form D, which showed adistinct Raman Spectrum and XRPD pattern. The phase transition by DSCwas much higher than the other forms and shows the highest stability ofthe four polymorphs identified. Form D can be directly prepared asfollows: A 250-mL three neck RBF equipped with a stir bar was chargedwith compound (I) (6.01 g, 11 mmol) and fumaric acid (1.41 g, 12 mmol).Acetone (50 mL) was added and the slurry was heated to 50° C. until thesolution became clear. Precipitation was observed after 10 min, andstirring was continued for an additional 30 min MTBE (25 mL) was addedand the solution was cooled to rt and stirred overnight. The solids werefiltered and dried under vacuum at 60° C. for 2 d to give the titlecompound (I) as a white solid (6.65 g, 91%).

1H NMR (400 MHz, CD₃OD) δ 8.00 (d, J=8.4 Hz, 1H), 7.68 (d, J=8.0 Hz,2H), 7.50-7.45 (m, 5H), 7.03 (d, J=8.1 Hz, 1H), 6.83 (d, J=8.4 Hz, 1H),6.73 (s, 2H), 6.60 (dd, J=8.4, 2.5 Hz, 1H), 5.58 (s, 1H), 4.00 (s, 2H),3.82-3.78 (m, 2H), 3.36 (t, J=8.4 Hz, 1H), 3.26 (s, 3H), 3.13-3.10 (m,2H), 2.34 (t, J=11.4 Hz, 2H), 2.25-2.16 (m, 2H), 1.01 (d, J=6.0 Hz, 6H).

TABLE 4 XRPD of Form D Rel. 2θ angle Int. (%) 9.59 97.3 11.69 39.2 12.0353.4 12.83 100.0 15.95 92.6 16.55 47.2 16.93 73.3 18.13 47.8 19.23 59.019.81 55.4 20.67 59.1 20.79 61.9 21.23 71.5 21.49 66.6 21.97 97.2 22.5342.1 23.97 53.9 24.03 46.2 26.03 32.5 29.75 41.6

The fumaric acid salt can be generated as form D under variousconditions. The crystal form can be generated through seeding withcrystals of form B or D as shown in Table 5. The salt can becrystallized directly by dissolving the parent compound (I) in polarsolvents such as ethyl acetate, acetone or ethanol and deliveringfumaric acid in polar solvents such as methanol, ethanol, THF andisopropanol, as shown in Table 6. Yields are generally improved when anantisolvent, such as MTBE, is added. Conditions that do not give form Dinclude when the compound (I) is dissolved into less polar solvents suchas acetonitrile, 2-methyltetrahydrofuran and methylisobutyl ketone.Also, addition of hexane as an antisolvent does not promote crystal formD.

The preferred Form D can also be generated by dissolving compound (I)into an appropriate solvent, such as methanol, ethanol, THF or acetoneand delivering fumaric acid directly as a solid, as seen in Table 7.Yields are improved when an antisolvent such as MTBE or IBAc is added.Form D is not formed by this method when the primary solvent is lesspolar, as is the case with ethyl acetate and MTBE.

A 2:1 compound (I)/fumaric acid crystalline salt could not be generated.Two equivalents of compound (I) was dissolved into a primary solventsuch as EtOAc, EtOH, THF, IPA and one equivalent of fumaric acid wasadded as a solid, or in a solution with IPA or EtOH. The resultantsolutions were heated to 50° C. for 30 min and cooled to rt. MTBE wasadded and the mixture was slurried for 24 h. Characterization of boththe solid and filtrate by ¹H NMR revealed only 1:1 compound (I)/fumaratesalt was obtained.

TABLE 5 Crystallization of fumarate salt with seeding Primary DeliverySeed Yield Solvent Solvent Equivalents Temp (° C.) addition (%) FormEtOAc EtOH 1.05 50 B 77 D EtOAc EtOH 1.05 50 D 67 D

TABLE 6 Crystallization of fumarate salt Primary Delivery Equiva- Anti-Yield Solvent Solvent lents Temp (° C.) solvent (%) Form EtOAc MeOH 1.0550 MTBE 54 D EtOAc EtOH 1.05 50 MTBE 51 D EtOAc THF 1.05 50 MTBE 53 DEtOAc IPA 1.05 50 MTBE 50 D EtOAc THF 1.05 50 Hexane — — 2-MeTHF MeOH1.05 50 MTBE — — 2-MeTHF EtOH 1.05 50 MTBE — — 2-MeTHF THF 1.05 50 MTBE— — Acetone MeOH 1.05 50 MTBE 75 D Acetone EtOH 1.05 50 No 40 D AcetoneTHF 1.05 50 MTBE 72 D Acetone THF 1.05 50 Hexane — — MIBK THF 1.05 50MTBE — — EtOH THF 1.05 50 MTBE 69 D EtOH THF 1.05 50 Hexane — — THF THF1.05 50 Hexane — —

TABLE 7 Crystallization with fumaric acid added as solid Primary SolventEquivalents Temp (° C.) Antisolvent Yield (%) Form MeOH 1.01 50 No 43 DEtOH 1.14 50 No 61 D EtOH 1.05 50 MTBE 74 D EtOH 1.05 50 IBAc 74 D THF1.01 50 MTBE 64 D Acetone 0.99 50 No 53 D Acetone 1.05 50 MTBE 82 DAcetone 1.05 50 IBAc 81 D EtOAc 1.01 50 No — — MTBE 1.01 50 No — —Rescoring of the Combinatorial Salt Screen Results

From the initial combinatorial process, only the fumarate salt wasidentified as crystalline. The results from the high throughput screenwere reevaluated and new scores were generated that were independent ofthe solvent for each counterion. This rescoring proceeded for thedifferent salts by combining the previous scores (visual inspection ofsolid formation, birefringence, uniqueness by Raman spectrum andthreshold solubility) over all the solvents and summing them up for boththe master and evaporation plates.

The two highest rescored acids, maleic and methane sulfonic acid, werefurther evaluated under additional solvent and antisolvent conditions.Salts obtained from methane sulfonic acid exhibited hygroscopicity.However, a crystalline salt with maleic acid was identified. Thepreparation and characterization of a maleate salt of compound A isdescribed below.

Example 7 Preparation of Form A of 1:1 Compound (I) Maleate

A 250-mL three neck round bottom flask equipped with a stir bar wascharged with compound (I) (4.96 g, 9.3 mmol). Acetone (55 mL) was addedand heated to 50° C. Maleic acid (20 mL of 0.5 M solution in acetone)was added resulting in a clear solution which became turbid after 1 min.The solution was cooled to rt and stirred for 24 h as a thick slurry.The solids were filtered, washed with MTBE and dried for 30 h at 60° C.under vacuum to give a white solid (5.52 g, 91%). The salt wascharacterized as a crystalline by XRPD and denoted form A.

¹H NMR (CD₃OD) δ: 8.04 (d, J=8.5 Hz, 1H), 7.77 (d, J=8.0 Hz, 2H),7.45-7.61 (m, 5H), 7.07 (d, J=8.8 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 6.62(dd, J=8.5, 2.5 Hz, 1H), 6.27 (s, 2H), 5.59 (d, J=2.5 Hz, 1H), 4.30 (s,2H), 3.86-3.82 (m, 2H), 3.34-3.43 (m, 2H), 3.27 (s, 3H), 2.74-2.68 (m,2H), 2.32-2.10 (m, 2H), 1.23 (d, J=6.3 Hz, 6H).

TABLE 8 XRPD of Maleate Form A 2θ angle rel. intensity 5.45 28.2 5.7554.4 10.83 70.9 11.49 111.9 12.37 79.3 12.59 103.2 14.05 38.1 14.91 96.115.07 100.0 16.73 29.6 17.05 75.9 17.83 20.6 18.55 51.7 19.51 34.8 19.9130.7 21.89 25.9 22.15 34.6 23.03 39.4 23.31 45.3 23.51 56.1 23.89 33.326.09 60.3

Maleate form A can be generated from a polar solvent, such as EtOH,acetone, isopropyl acetate, ethyl acetate, isopropanol or THF with orwithout addition of a non-polar antisolvent, such as MTBE. A 2:1compound (I)/maleic acid crystalline salt could not be generated.

Preparation of Amorphous Salts of Compound (I) Example 8 Preparation ofHCl Salt of Compound (I)

Compound (I) (6.7 g, 12.5 mmol) was dissolved into THF (25 mL) and 1MHCl in ether (13.8 mL, 13.8 mmol) was added at rt and the solutiondiluted with ether (200 mL). The mixture was stored at rt for 1 h, andthe resulting solid was filtered. The solid forms a gel upon standingand was dissolved into water (100 mL) and freeze dried to a yellowpowder (5.8 g, 81%).

¹H NMR (400 MHz, CD₃OD) δ 7.75 (d, J=8.4 Hz, 1H), 7.57 (d, J=8.0 Hz,2H), 7.46 (d, J=8.0 Hz, 2H), 7.42 (s, 1H), 7.35 (d, J=16.8 Hz, 1H), 7.30(d, J=16.4 Hz, 1H), 6.82 (d, J=9.2 Hz, 1H), 6.80 (d, J=8.8 Hz, 1H), 6.51(d, J=8.4 Hz, 1H), 5.19 (s, 1H), 4.28 (s, 2H), 3.92-3.80 (m, 2H),3.40-3.30 (m, 2H), 3.27 (t, J=8.4 Hz, 1H), 3.15 (s, 3H), 2.70 (t, J=11.4Hz, 2H), 2.15-2.05 (m, 2H), 1.18 (d, J=6.0 Hz, 6H).

Example 9 Preparation of Phosphate Salt of Compound (I)

Compound (I) (148 mg, 0.27 mmol) was dissolved into EtOAc (0.5 mL) and0.5M phosphoric acid in EtOAc (0.58 mL, 0.28 mmol) was added at 50° C.and the solution was stirred for 15 min and cooled to rt. The solidswere filtered and dried for 4 d at 60° C. to give the title compound(I)s a white solid (149 mg, 85%).

¹H NMR (CD₃OD) δ 8.00 (d, J=8.5 Hz, 1H), 7.72 (d, J=8.2 Hz, 2H),7.49-7.58 (m, 5H), 7.04 (d, J=8.0 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 6.61(dd, J=8.5, 2.5 Hz, 1H), 5.58 (d, J=2.5 Hz, 1H), 4.21 (s, 2H), 3.97-3.89(m, 2H), 3.34-3.39 (m, 1H), 3.26 (s, 3H), 2.55 (t, J=11.8 Hz, 2H),2.16-2.26 (m, 2H), 1.19 (d, J=6.0 Hz, 6H).

Solubility Test and Pharmacokinetic Analyses

Methods:

A single oral dose in solution and as powder in capsule of the HCl saltof (I), fumarate form D and maleate form A were administered to femaleSprague-Dawley rats at 5 mg/kg. Blood samples were collected in thepresence of lithium heparin, and were centrifuged to generate plasma.The plasma was then analyzed for compound (I) plasma levels by LC/MS.

Preparation of Test Article for Capsule Dosing in Rats:

All animals involved in the dosing schedule were weighed and assignednumbers. All capsules (Size 9 porcine hard gelatin capsules, Torpac)corresponding with each animal to be dosed were carefully numbered witha fine tip Sharpie. A small plastic weighing dish was placed on thescale, with one capsule lid placed on the dish and the base of thecapsule loader with the other part of the capsule was loaded in thereservoir. The balance was tared. A funnel was placed on top of thecapsule loading apparatus and the total mass was carefully noted. Apiece of 4″×4″ wax paper was folded in half creating a crease down themedian. A small amount of finely ground compound (Ground the compound ifrequired using mortar and pestle) was deposited in the crease of the waxpaper and refolded. The folded wax paper was gently angled over thecapsule loader funnel apparatus. A fine scapula was used to tease anamount of compound powder out of the crease onto the funnel allowing itto trickle into the loading bay and ultimately the capsule. Thedifference in mass from the total mass was noted. The capsule loadingfunnel was removed and the balance door closed to ascertain the absoluteweight of compound inside the capsule. The funnel was returned to thecapsule loading apparatus and loading continued until the desired amountof drug was inside the capsule. Note: When adding minute quantities tothe capsule, simply brushed fine powered drug that settled on the funnelinto the loading column. The funnel was removed and the weight wasrecorded. Calculated the amount of actual drug by multiplying weighedmaterial by the bioequivalence ratio. The amount of actual drug wasrecorded. The capsule lid on the weighing dish was used to close thecapsule tightly until it clicked into place.

All animals (n=3/group) were dosed orally in a volume of 5 mL/kg.Following dosing, each rat was bled at each of the designated timepoints. For control animals, blood was collected by the same procedure.Blood was collected from the lateral saphenous vein. Blood aliquots (˜50μL) were collected in tubes coated with lithium heparin, mixed gently,then kept on ice and centrifuged at 2,500×g for 15 minutes at RT, within1 hour of collection. The plasma layer was collected, kept on ice andfinally maintained frozen at −80° C. until further processing.

Bioanalytical Methods

Bioanalytical quantitation using HPLC/tandem quadruple mass spectrometry(HPLC-MS) was performed. Plasma concentrations and T_(1/2) werecalculated and reported.

Plasma Assay:

A 5 mg/mL standard solution in DMSO was diluted 100 fold andsubsequently serial diluted in 50% DMSO. Aliquots (2 μL) of the serialdilutions were mixed with 18 μL of control plasma (20 μL total) for useas a standard curve. Plasma samples (20 μL) and standard samples werethen diluted 5× with ice cold acetonitrile containing 100 ng/mLverapamil as internal standard (80% (v/v) acetonitrile). Acetonitrileprecipitated samples and standards were filtered through 0.22 μmmembranes in a 96-well format. Filtrates were then diluted with water to30% acetonitrile.

Dose Assay:

Dosing solution (100 μL) was diluted with DMSO (900 μL) to ensure samplehomogeneity. Dilution of the resulting solution into 30% acetonitrile(containing internal standard) was then performed in triplicate to bringthe nominal concentration to less than 500 ng/mL, appropriate for LC-MSanalysis. Serial dilution from a 5 mg/mL DMSO stock, into 30%acetonitrile (containing internal standard) provided a suitable standardcurve. Samples and standards (10 μL) were injected into the LC-MSsystem, as described below. Concentrations of dose solutions werereported in mg/mL.

LC-MS Analysis:

LC: 10 μL of each sample and standard were injected onto a WatersAcquity CSH 1.7 μm 2.1×100 mm column at 0.6 mL/min by an Acquity UPLC.The C18 column was equilibrated at 10% acetonitrile. Compounds wereeluted with a gradient to 99% acetonitrile. All mobile phases contained0.1% (v/v) formic acid.

Chromatographic Elution:

t (min) % B 0 5 0.75 5 1 20 4.5 99.9 5 99.9 5.4 5 6 5

MS: Column eluent was analyzed by electrospray ionization into a tandemquadrupole mass spec system (Waters Xevo TQ). Eluent composition wasanalyzed for three ion-pairs specific for the internal standard andthree ion-pairs specific for the analyte.

Pharmacokinetic Analyses

Experimental samples were compared with standard curve samples todetermine compound concentrations. Average compound concentrations (inμg/mL+/−standard deviation) were reported for each time-point. Limit ofDetection (LLOQ) was reported as the lowest standard curve sampledemonstrating a deviation of less than 20% of nominal concentration. PKanalysis was performed by the Excel plugin PKfit; C_(max) weredetermined as the maximum average concentration observed at a given timepoint; the area under the curve (AUC) was reported for t₀ to t_(last)hours. Plasma half-lives were reported when a minimum of 3 terminaltime-points demonstrated first order elimination with an r²>0.8.

As shown in Table 9, crystalline salts of compound (I) were shown to beless soluble in deionized water compared to the amorphous phosphate andHCl salts. In many reported cases, the increased solubility of theamorphous salts results in an increase in plasma concentrations relativeto a more stable crystalline form (Hancock and Parks (2000) Pharm. Res.17: 397-404; Pudipeddi and Serajuddin (2005) J. Pharm. Sci. 94: 929-39).However, when the plasma concentration-time profiles and pharmacokineticparameters of amorphous and crystalline salts were compared followingoral dosing as powder in capsule (PIC) to female Sprague-Dawley rats,the difference in pharmacokinetic parameters was minimal (Table 10).

TABLE 9 Characterization summary of compound (I) Salts CompoundGravimetric Solubility in (I):acid TGA Loss Moisture DI water CounterionXRPD ratio DSC (° C.) (wt %) Sorption (μg/mL) Fumarate A 1.0:0.9 112,158  7.2 (45-160° C.) Fumarate B 1.0:1.0 58, 162 2.6 (40-120° C.) 3.7 wt% 60% RH 10.5 wt % 90% RH Fumarate C 1.0:1.1 62, 156 2.6 (30-100° C.)625 Fumarate D 1.0:1.0 219 0.9 (30-100° C.) 2.1 wt % 60% RH 170 4.0 wt %90% RH Maleate A  1.0:0.95 219 No loss 1.3 wt %, 60% RH observed 2.2 wt%, 90% RH until melt Phosphate Amorphous 1.0:1.1 83, 179 1.9 6.1 wt %60% RH ≧1 × 10⁵ 10.9 wt % 90% RH HCl Amorphous 2.3 (30-150° C.) ≧1 × 10⁵

TABLE 10 Pharmacokinetic Parameters after PO Administration of compound(I) Salts, Powder in Capsule, to Sprague-Dawley Rats Fumarate MaleateSalt form HCl (form D) (form A) Oral Dose (mg/kg) 5.0 5.0 5.0 Cmax(ng/mL) 250 270 200 AUC_(0-tlast) (ng · h/mL) 2400 2780 1500

What is claimed is:
 1. A fumarate salt of compound (I) represented bythe following structural formula:

wherein the molar ratio between compound (I) and fumaric acid is 1:1,wherein at least 90% by weight of the fumarate salt is singlecrystalline form A, characterized by an X-ray powder diffraction patternwhich comprises peaks at 8.2°, 9.7°, 16.7°, and 20.1°±0.2 in 2θ.
 2. Thefumarate salt of claim 1, wherein at least 99% by weight of the salt isin a single crystalline form.
 3. The fumarate salt of claim 1, whereinat least 90% by weight of the fumarate salt is single crystalline formA, characterized by differential scanning calorimeter (DSC) peak phasetransition temperatures of 112° C. and 158° C.
 4. The fumarate salt ofclaim 1, wherein at least 90% by weight of the fumarate salt is singlecrystalline form A, characterized by an X-ray powder diffraction patternwhich comprises peaks at 8.2°, 9.7°, 10.7°, 11.5°, 13.6°, 14.9°, 16.7°,18.1°, 18.8°, 20.1°, 23.5°, and 24.5°±0.2 in 2θ.
 5. A pharmaceuticalcomposition comprising the fumarate salt of claim 1, and apharmaceutically acceptable carrier or diluent.
 6. A fumarate salt ofcompound (I) represented by the following structural formula:

wherein the molar ratio between compound (I) and fumaric acid is 1:1,wherein at least 90% by weight of the fumarate salt is singlecrystalline form B, characterized by an X-ray powder diffraction patternwhich comprises peaks at 11.9°, 14.9°, 18.7°, and 21.5°±0.2 in 2θ. 7.The fumarate salt of claim 6, wherein at least 99% by weight of the saltis in a single crystalline form.
 8. The fumarate salt of claim 6,wherein at least 90% by weight of the fumarate salt is singlecrystalline form B, characterized by differential scanning calorimeter(DSC) peak phase transition temperatures of 58 ° C. and 162° C.
 9. Thefumarate salt of claim 6, wherein at least 90% by weight of the fumaratesalt is single crystalline form B, characterized by an X-ray powderdiffraction pattern which comprises peaks at 5.5°, 5.9°, 11.9°, 14.9°,16.7°, 17.4°, 18.7°, 21.5°, and 23.4°±0.2 in 2θ.
 10. A pharmaceuticalcomposition comprising the fumarate salt of claim 6, and apharmaceutically acceptable carrier or diluent.
 11. A fumarate salt ofcompound (I) represented by the following structural formula:

wherein the molar ratio between compound (I) and fumaric acid is 1:1,wherein at least 75-80% by weight of the fumarate salt is singlecrystalline form C, characterized by an X-ray powder diffraction patternwhich comprises peaks at 9.8°, 16.8°, 16.9°, 19.9°, and 23.5°±0.2 in 2θ.12. The fumarate salt of claim 11, wherein at least 99% by weight of thesalt is in a single crystalline form.
 13. The fumarate salt of claim 11,wherein at least 90% by weight of the fumarate salt is singlecrystalline form C, characterized by differential scanning calorimeter(DSC) peak phase transition temperatures of 62° C. and 156° C.
 14. Thefumarate salt of claim 11, wherein at least 75-80% by weight of thefumarate salt is single crystalline form C, characterized by an X-raypowder diffraction pattern which comprises peaks at 9.7°, 9.8°, 11.7°,15.1°, 16.8°, 16.9°, 19.9°, 23.5°, and 23.7°±0.2 in 2θ.
 15. Apharmaceutical composition comprising the fumarate salt of claim 11, anda pharmaceutically acceptable carrier or diluent.
 16. A fumarate salt ofcompound (I) represented by the following structural formula:

wherein the molar ratio between compound (I) and fumaric acid is 1:1,wherein at least 90% by weight of the fumarate salt is singlecrystalline form D, characterized by an X-ray powder diffraction patternwhich comprises peaks at 9.6°, 12.8°, 16.0°, 16.9°, 21.2°, and 22.0°±0.2in 2θ.
 17. The fumarate salt of claim 16, wherein at least 99% by weightof the salt is in a single crystalline form.
 18. The fumarate salt ofclaim 16, wherein at least 90% by weight of the fumarate salt is singlecrystalline form D, characterized by differential scanning calorimeter(DSC) peak phase transition temperature of 219° C.
 19. The fumarate saltof claim 16, wherein at least 90% by weight of the fumarate salt issingle crystalline form D, characterized by an X-ray powder diffractionpattern which comprises peaks at 9.6°, 11.7°, 12.0°, 12.8°, 16.0°,16.6°, 16.9°, 18.1°, 19.2°, 19.8°, 20.7°, °.8°, 21.2°, 21.5°, 22.0°,22.5°, 24.0°, 26.0°, and 29.8°±0.2 in 2θ.
 20. A pharmaceuticalcomposition comprising the fumarate salt of claim 16, and apharmaceutically acceptable carrier or diluent.